The present invention relates to vascular endothelial growth factor (VEGF). More particularly, the invention relates to antagonists of VEGF and use of those antagonists in the treatment of disorders that are associated with VEGF.
Blood vessels are the means by which oxygen and nutrients are supplied to living tissues and waste products are removed from living tissue. Angiogenesis refers to the process by which new blood vessels are formed. See, for example, the review by Folkman and Shing, J. Biol. Chem. 267, 10931-10934 (1992), Dvorak, et al., J. Exp. Med., 174, 1275-1278 (1991). Thus, where appropriate, angiogenesis is a critical biological process. It is essential in reproduction, development and wound repair. However, inappropriate angiogenesis can have severe negative consequences. For example, it is only after many solid tumors are vascularized as a result of angiogenesis that the tumors have a sufficient supply of oxygen and nutrients that permit it to grow rapidly and metastasize. Because maintaining the rate of angiogenesis in its proper equilibrium is so critical to a range of functions, it must be carefully regulated in order to maintain health. The angiogenesis process is believed to begin with the degradation of the basement membrane by proteases secreted from endothelial cells (EC) activated by mitogens such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The cells migrate and proliferate, leading to the formation of solid endothelial cell sprouts into the stromal space, then, vascular loops are formed and capillary tubes develop with formation of tight junctions and deposition of new basement membrane.
In adults, the proliferation rate of endothelial cells is typically low compared to other cell types in the body. The turnover time of these cells can exceed one thousand days. Physiological exceptions in which angiogenesis results in rapid proliferation typically occurs under tight regulation, such as found in the female reproduction system and during wound healing.
The rate of angiogenesis involves a change in the local equilibrium between positive and negative regulators of the growth of microvessels. The therapeutic implications of angiogenic growth factors were first described by Folkman and colleagues over two decades ago (Folkman, N. Engl. J. Med., 285:1182-1186 (1971)). Abnormal angiogenesis occurs when the body loses at least some control of angiogenesis, resulting in either excessive or insufficient blood vessel growth. For instance, conditions such as ulcers, strokes, and heart attacks may result from the absence of angiogenesis normally required for natural healing. In contrast, excessive blood vessel proliferation can result in tumor growth, tumor spread, blindness, psoriasis and rheumatoid arthritis.
Thus, there are instances where a greater degree of angiogenesis is desirablexe2x80x94increasing blood circulation, wound healing, and ulcer healing. For example, recent investigations have established the feasibility of using recombinant angiogenic growth factors, such as fibroblast growth factor (FGF) family (Yanagisawa-Miwa, et al., Science, 257:1401-1403 (1992) and Baffour, et al., J Vasc Surg, 16:181-91 (1992)), endothelial cell growth factor (ECGF)(Pu, et al., J Surg Res. 54:575-83 (1993)), and more recently, vascular endothelial growth factor (VEGF) to expedite and/or augment collateral artery development in animal models of myocardial and hindlimb ischemia (Takeshita, et al., Circulation, 90:228-234 (1994) and Takeshita, et al., J Clin Invest. 93:662-70 (1994)).
Conversely, there are instances, where inhibition of angiogenesis is desirable. For example, many diseases are driven by persistent unregulated angiogenesis, also sometimes referred to as xe2x80x9cneovascularization.xe2x80x9d In arthritis, new capillary blood vessels invade the joint and destroy cartilage. In diabetes, new capillaries invade the vitreous, bleed, and cause blindness. Ocular neovascularization is the most common cause of blindness. Tumor growth and metastasis are angiogenesis-dependent. A tumor must continuously stimulate the growth of new capillary blood vessels for the tumor itself to grow.
There is mounting evidence that VEGF may be a major regulator of angiogenesis (reviewed in Ferrara, et al., Endocr. Rev., 13, 18-32 (1992); Klagsbrun, et al., Curr. Biol., 3, 699-702 (1993); Klagsbrun, et al., Ferrara, et al., Biochem. Biophys. Res. Commun., 161, 851-858 (1989) ). VEGF was initially purified from the conditioned media of folliculostellate cells (Ferrara, et al., Biochem. Biopsy. Res. Commun., 161, 851-858 (1989)) and from a variety of tumor cell lines (Myoken, et al., Proc. Nat. Acad. Sci. USA, 88:5819-5823 (1991); Plouet, et al., EMBO. J., 8:3801-3806 (1991)). VEGF was found to be identical to vascular permeability factor, a regulator of blood vessel permeability that was purified from the conditioned medium of U937 cells at the same time (Keck, et al., Science, 246:1309-1312 (1989)). VEGF is a specific mitogen for endothelial cells (EC) in vitro and a potent angiogenic factor in vivo. The expression of VEGF is up-regulated in tissue undergoing vascularization during embryogenesis and the female reproductive cycle (Brier, et al., Development, 114:521-532 (1992); Shweiki, et al., J. Clin. Invest., 91:2235-2243 (1993)). High levels of VEGF are expressed in various types of tumors, but not in normal tissue, in response to tumor-induced hypoxia (Shweiki, et al., Nature 359:843-846 (1992); Dvorak et al., J. Exp. Med., 174:1275-1278 (1991); Plate, et al., Cancer Res., 53:5822-5827; Ikea, et al., J. Biol. Chem., 270:19761-19766 (1986)). Treatment of tumors with monoclonal antibodies directed against VEGF resulted in a dramatic reduction in tumor mass due to the suppression of tumor angiogenesis (Kim, et al., Nature, 382:841-844 (1993)). VEGF appears to play a principle role in many pathological states and processes related to neovascularization. Regulation of VEGF expression in affected tissues could therefore be key in treatment or prevention of VEGF induced neovascularization/angiogenesis.
VEGF is a secreted 40-45K homodimer (Tischer E. et. al., J. Biol. Chem. 266: 11947-11954 (1991). It is a member of an expanding family that includes placenta-derived growth factor (PIGF), VEGF-B, VEGF-C, VEGF-D and VEGF-E (Olofsson et. al., Proc. Natl. Acad. Sci. USA 93:2576-2581 (1996), Joukov et. al., EMBO J. 15:290-298 (1996). Achen et. al., Proc. Natl.Acad. Sci.USA 95:548-553 (1998). Ogawa et. al., J. Biol. Chem. 273: 31273-31282 (1998)). VEGF exists in a number of different isoforms that are produced by alternative splicing from a single gene containing eight exons (Ferrara, et al., Endocr. Rev., 13:18-32 (1992); Tischer, et al., J. Biol. Chem., 806:11947-11954 (1991); Ferrara, et al., Trends Cardio Med., 3:244-250 (1993); Polterak, et al., J. Biol. Chem., 272:7151-7158 (1997)). Human VEGF isoforms consists of monomers of 121, 145, 165, 189, and 206 amino acids, each capable of making an active homodimer (Polterak et al., J. Biol. Chem, 272:7151-7158 (1997); Houck, et al., Mol. Endocrinol., 8:1806-1814 (1991)). The VEGF121 and VEGF165 isoforms are the most abundant. VEGF121 is the only VEGF isoforms that does not bind to heparin and is totally secreted into the culture medium. VEGF165 is functionally different than VEGF121 in that it binds to heparin and cell surface heparin sulfate proteoglycans (HSPGs) and is only partially released into the culture medium (Houck, et al., J. Biol. Chem., 247:28031-28037 (1992); Park, et al., Mol. Biol. Chem., 4:1317-1326 (1993)). The remaining isoforms are entirely associated with cell surface and extracellular matrix HSPGs (Houck, et al., J. Biol. Chem., 247:28031-28037 (1992); Park, et al., Mol. Biol. Chem., 4:1317-1326 (1993)).
VEGF receptor tyrosine kinases, KDR/Flk-1 and/or Flt-1, are mostly expressed by EC (Terman, et al., Biochem. Biophys. Res. Commun., 187:1579-1586 (1992); Shibuya, et al., Oncogene, 5:519-524 (1990); De Vries, et al., Science, 265:989-991 (1992); Gitay-Goran, et al., J. Biol. Chem., 287:6003-6096 (1992); Jakeman, et al., J. Clin. Invest., 89:244-253 (1992)). It appears that VEGF activities such as mitogenicity, chemotaxis, and induction of morphological changes are mediated by KDR/Flk-1 but not Flt-1, even though both receptors undergo phosphorylation upon binding of VEGF (Millauer, et al., Cell, 72:835-846 (1993): Waltenberger, et al., J. Biol. Chem., 269:26988-26995 (1994); Seetharam, et al., Oncogene, 10:135-147 (1995): Yoshida, et al., Growth Factors, 7:131-138 (1996)). Recently, Sokeret et al., identified a new VEGF receptor which is expressed on EC and various tumor-derived cell lines such as breast cancer-derived MDA-MB-231 (231) cells (Soker, et al., J. Biol. Chem., 271:5761-5767 (1996)). This receptor requires the VEGF isoform to contain the portion encoded by exon 7. For example, although both VEGF121 and VEGF165R bind to KDR/Flk-1 and Flt-1, only VEGF165 binds to the new receptor. Thus, this is an isoform-specific receptor and has been named the VEGF165 receptor (VEGF165R). It will also bind the 189 and 206 isoforms. In structure-function analysis, it was shown directly that VEGF165 binds to VEGF165R via its exon 7-encoded domain which is absent in VEGF121 (Soker, et al., J. Biol. Chem., 271:5761-5767 (1996)). However, the function of the receptor was unclear.
The current treatment of angiogenic diseases is inadequate. Agents which prevent continued angiogenesis, e.g., drugs (TNP-470), monoclonal antibodies, antisense nucleic acids and proteins (angiostatin and endostatin) are currently being tested. See, Battegay, J. Mol. Med., 73, 333-346 (1995); Hanahan et al., Cell, 86, 353-364 (1996); Folkman, N. Engl. J. Med., 333, 1757-1763 (1995). Although preliminary results with the antiangiogenic proteins are promising, they are relatively large in size and thus difficult to use and produce. Moreover, proteins are subject to enzymatic degradation. Thus, new agents that inhibit angiogenesis are needed. New antiangiogenic proteins or peptides that show improvement in size, ease of production, stability and/or potency would be desirable.
We have discovered that a portion of the seventh exon of VEGF165 acts as an antagonist to all VEGF isoforms, which is surprising since not all forms of VEGF have exon 7. For example, we have prepared a glutathione S-transferase (GST) fusion protein containing a peptide corresponding to the 44 amino acids encoded by exon 7 and the first cystein of the peptide encoded by exon 8 (amino acids 116-160 of VEGF165 (SEQ ID NO: 1)). This fusion protein inhibited the binding of 125I-VEGF165 to receptors on human umbilical cord vein-derived EC (HUVEC) and on 231 cells. The inhibitory activity was localized to the C-terminal portion of the exon 7-encoded domain (amino acids 22-44). Furthermore, the fusion protein inhibited VEGF-induced proliferation of HUVEC. The fusion protein also inhibits VEGF121-induced mitogenicity, which was an unexpected result considering that VEGF121 does not contain exon 7. Thus, the polypeptides of the present invention are antagonists against the major isoforms of VEGF and can be used to treat diseases and conditions associated with VEGF-induced neovascularization or angiogenesis.
In addition, while not wishing to be bound by theory, it is believed that VEGF is directly associated with a number of cancers expressing the VEGF165R/NP-1 (Soker, et al., Cell 92, 735-745 (1998)), and that inhibition of VEGF binding to this receptor can be used to treat such cancers.
The present invention provides a polypeptide having a portion of SEQ ID NO:1 having VEGF antagonist activity as determined, for example, by the human umbilical vein endothelial cell (HUVEC) proliferation assay using VEGF165 as set forth below in the Examples. Preferably, the portion has at least a 25% reduction in HUVEC proliferation, more preferably a 50% reduction, even more preferably a 75% reduction, most preferably a 95% reduction. Preferably, the portion has an even number of cysteine residues.
VEGF antagonist activity may also be determined by inhibition of binding of labeled VEGF165 to VEGF165R as disclosed in Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)) and forth below in the Examples. Preferably, the portion inhibits binding by at least 25%, more preferably 50%, most preferably 75%.
The present invention further provides polypeptides comprising SEQ ID NO: 2 (CSCKNTDSRCKARQLELNERTCRC) or a portion thereof having VEGF antagonist activity as determined, for example, by the human umbilical vein endothelial cell (HUVEC) proliferation assay using VEGF165 as set forth below in the Examples. Preferably, the portion has at least a 25% reduction in HUVEC proliferation, more preferably a 50% reduction, even more preferably a 75% reduction, most preferably a 95% reduction. Preferably, the portion has an even number of cysteine residues.
One preferred polypeptide of the present invention has the structure of the following formula (I):
(X1-(CSCKNTDSRCKARQLELNERT(SEQ ID NO:3))-X2)xe2x80x83xe2x80x83I
wherein X1 is H, or any portion of amino acids 2-21 of SEQ ID NO: 1. For example, amino acid 3-21, 4-21, 5-21, 6-21, etc. of SEQ ID NO: 1. And X2 is H or C, CR, RC or CRC. The polypeptides of formula (I) have VEGF antagonist activity as determined, for example, by the human umbilical vein endothelial cell (HUVEC) proliferation assay using VEGF165 as set forth below in the Examples. Preferably, the polypeptide has at least a 25% reduction in HUVEC proliferation, more preferably a 50% reduction, even more preferably a 75% reduction, most preferably a 95% reduction. Preferably, the polypeptide has an even number of cysteine residues. The polypeptides of formula (I) include analogs.
xe2x80x9cAnalogsxe2x80x9d refers to a polypeptide differing from the sequence of one of the peptides of the invention but which still exhibits at least 50% of the VEGF antagonist activity of the polypeptide of SEQ ID NO: 2 in the human umbilical vein endothelial cell (HUVEC) proliferation assay using VEGF165 as set forth below in the Examples. Preferably, the analog exhibits 75% of the VEGF antagonist activity of the polypeptide of SEQ ID NO: 2, most preferably 95%. The differences are preferably conservative amino acid substitutions, in which an amino acid is replaced with another naturally occurring amino acid of similar character. For example, the following substitutions are considered xe2x80x9cconservativexe2x80x9d: Gly  Ala; Val  Ile; Asp  Glu; Lys  Arg; Asn  Gln; and Phe  Trp  Tyr. Nonconservative changes are generally substitutions of one of the above amino acids with an amino acid from a different group (e.g., substituting Asn for Glu), or substituting Cys, Met, His, or Pro for any of the above amino acids.
In preferred forms, the polypeptides of the present invention are part of a fusion protein or conjugated to a moiety to enhance purification, increase stability and/or to provide a biological activity.
In another embodiment, the polypeptides of the present invention, either alone, or as part of a fusion protein, are used to target cells expressing the VEGF165R/NP-1. This targeting can be used for diagnostic as well as therapeutic applications. For example, for diagnostic purposes the polypeptide is radiolabeled and used to detect cells expressing the VEGF165R/NP-1. We have discovered that expression of the receptor has a high correlation to disease state in a number of cancers, such as prostate and breast, particularly metastatic cancers. Accordingly, in a further embodiment, the polypeptide can be used in a prognostic manner for particular cancers.
For therapeutic applications, the polypeptide can be used to deliver agents to cells expressing the VEGF165R/NP-1. For example, the polypeptides can be used as carriers to deliver a desired chemical or cytotoxic moiety to the cells. The cytotoxic moiety may be a cytotoxic drug or an enzymatically active toxin of bacteria, fungal or plant origin, or an enzymatically active polypeptide chain or fragment (xe2x80x9cA chainxe2x80x9d) of such a toxin. Enzymatically active toxins and fragments thereof are preferred and are exemplified by diphtheria toxin A fragment, non-binding active fragments of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alphasarcin, certain Aleurites fordii proteins, certain Dianthin proteins, Phytolacca americana proteins (PAP, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin. Ricin A chain, Pseudomonas aeruginosa exotoxin A and PAP are preferred.
The invention further provides a method of treating a disease or disorder/condition associated with VEGF-induced neovascularization or angiogenesis. As used herein, the term xe2x80x9cneovascularizationxe2x80x9d refers to the growth of blood vessels and capillaries. Diseases, disorders, or conditions, associated with VEGF-induced neovascularization or angiogenesis, include, but are not limited to retinal neovascularization, hemagiomas, solid tumor growth, leukemia, metastasis, psoriasis, neovascular glaucoma, diabetic retinopathy, rheumatoid arthritis, osteoarthritis, endometriosis, mucular degeneration and retinopathy of prematurity (ROP).
In the methods of the present invention, a therapeutic amount of a polypeptide of the invention is administered to a host, e.g., human or other mammal, having a disease or condition, associated with VEGF or having a rumor expressing VEGF165R/NP-1. Methods for detecting the expression of VEGF165R/NP-1 are set forth in Soker, et al., Cell 92:735-745 (1998).
The invention also provides a composition comprising an effective amount of a polypeptide of the invention in combination with a pharmaceutically acceptable carrier.
Other aspects of the invention are disclosed infra.